The objective of this proposal is to uncover the in vivo mechanisms by which STING (Stimulator of interferon genes) mediates the mucosal vaccine adjuvant activity of cyclic di-GMP (CDG). Protective mucosal immune responses are most effectively induced by mucosal immunization. However, most of the currently approved human vaccines are administered systemically and generally fail to elicit effective mucosal immunity. Live attenuated mucosal vaccines present safety and acceptability issues while purified antigens are generally poor immunogenics when administered by the mucosal route. CDG exhibits potent mucosal immunogenicity thus, has been explored as a promising mucosal vaccine adjuvant. The mechanism by which CDG executes its mucosal adjuvant activity is unknown, which hinders the further development of CDG as an efficacious mucosal adjuvant. STING, also known as MPYS/MITA, is essential for type I IFN (IFN-I) production by cytosolic sensing of DNA. Recently, we showed that STING mediates IFN-I production by CDG. Subsequent structure studies found that the cytoplasmic tail of STING binds to CDG. It is proposed that STING is a direct sensor for CDG, leading to IFN-I production. However, the in vivo role of STING in CDG-induced vaccine adjuvant activity is not known. In this proposal, we found that STING-/- mice fail to generate antigen-specific antibody response after intranasal immunization of an antigen and CDG. Furthermore, the production of Th1/Th2/Th17 cytokines, proinflammatory cytokines and IFN-I are absent in CDG immunized STING-/- mice. Surprisingly, we found that IFN-I signaling is NOT required for the mucosal adjuvant activity of CDG because IFNAR1-/- mice have the same antigen-specific antibody response as the wild-type mice. This is distinct from STING-mediated DNA vaccine adjuvant activity, which requires IFN-I signaling. We propose two specific Aims to address the in vivo cellular and in vitro molecular mechanisms underlying STING-mediated CDG mucosal adjuvant activity. Aim 1: Dendritic Cells (DC) Expression of STING is essential for the mucosal vaccine adjuvant activity of CDG in vivo. We will use conditional STING-/- and conditional STINGwt/wt mice generated by us to determine the cellular mechanism of STING-mediated CDG adjuvant activity in vivo; Aim 2: Determine the IFN-I stimulation independent mechanism underlying STING-mediated DC activation by CDG. We found that CDG promotes STING-dependent but IFN-I signaling-independent DC maturation. We will use biochemical and molecular biological approaches to identify the proximal signaling transducer of STING that mediates this IFN-I stimulation-independent function. We will further verify its importance in the adjuvant activity of CDG in vivo using KO mice. Currently, there is no vaccine formulation containing a mucosal adjuvant approved for human use. The knowledge generated by our studies can help advance the development of CDG as an efficacious mucosal vaccine adjuvant for human use.